1. The relaxant effects of isoprenaline may result from activation of another beta-adrenoceptor subtype in addition to beta1 and beta2. This study evaluated the role of a third beta-adrenoceptor subtype, beta3, in beta-adrenoceptor-induced relaxation of rat thoracic aorta by isoprenaline. 2. Isoprenaline produced a concentration-dependent relaxation of phenylephrine pre-contracted rings of the thoracic aorta (pD2=7.46+/-0.15; Emax=85.9+/-3.4%), which was partially attenuated by endothelium removal (Emax=66.5+/-6.3%) and administration of the nitric oxide (NO) synthase inhibitor, L-NG-monomethyl arginine (L-NMMA) (Emax=61.3+/-7.9%). 3. In the presence of nadolol, a beta1- and beta2-adrenoceptor antagonist, isoprenaline-induced relaxation persisted (Emax=55.6+/-5.3%), but occurred at higher concentrations (pD2=6.71+/-0.10) than in the absence of nadolol and lasted longer. 4. Similar relaxant effects were obtained with two beta3-adrenoceptor agonists: SR 58611 (a preferential beta3-adrenoceptor agonist), and CGP 12177 (a partial beta3-adrenoceptor with beta1- and beta2-adrenoceptor antagonistic properties). SR 58611 caused concentration-dependent relaxation (pD2=5.24+/-0.07; Emax=59.5+/-3.7%), which was not modified by pre-treatment with nadolol but antagonized by SR 59230A, a beta3-adrenoceptor antagonist. The relaxation induced by SR 58611 was associated with a 1.7 fold increase in tissue cyclic GMP content. 5 Both relaxation and the cyclic GMP increase induced by SR 58611 were greatly reduced by endothelium removal and in the presence of L-NMMA. 6 We conclude that in the rat thoracic aorta, beta3-adrenoceptors are mainly located on endothelial cells, and act in conjuction with beta1- and beta2-adrenoceptors to mediate relaxation through activation of an NO synthase pathway and subsequent increase in cyclic GMP levels.